Publications by authors named "Yoshiteru Azuma"

27 Publications

  • Page 1 of 1

Carnitine supplementation prevents carnitine deficiency caused by pivalate-conjugated antibiotics in patients with epilepsy prescribed valproate.

Epilepsy Behav 2021 Apr 11;117:107883. Epub 2021 Mar 11.

Department of Pediatrics, Aichi Medical University, Japan.

We measured carnitine levels before and after pivalate-conjugated antibiotic (PCA) use in six patients with epilepsy who were prescribed valproate (VPA). Three of the patients were on carnitine supplementation when PCA use started. Serum FC levels were within the normal range (37.2-49.0 μmol/L) in all six patients before PCA use. After PCA use, the serum free carnitine (FC) levels remained within the normal range (48.0-68.2 μmol/L) in all three patients on carnitine supplementation, but were below the normal range (18.7-30.8 μmol/L) in the three patients not on carnitine supplementation. No remarkable changes in serum VPA levels, platelet count, amylase or ammonia level was evident in any patients in relation to PCA use. Carnitine deficiency due to PCA use was prevented by carnitine supplementation in patients with epilepsy who were taking VPA. Carnitine supplementation can support patients at risk of carnitine deficiency.
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http://dx.doi.org/10.1016/j.yebeh.2021.107883DOI Listing
April 2021

Hemizygous FLNA variant in West syndrome without periventricular nodular heterotopia.

Hum Genome Var 2020 Dec 3;7(1):43. Epub 2020 Dec 3.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa, Yokohama, 236-0004, Japan.

Pathogenic FLNA variants can be identified in patients with seizures accompanied by periventricular nodular heterotopia (PVNH). It is unusual to find FLNA aberrations in epileptic patients without PVNH on brain imaging. We report a boy with cryptogenic West syndrome followed by refractory seizures and psychomotor delay. We performed whole-exome sequencing and identified a de novo missense variant in FLNA. It is noteworthy that this patient showed no PVNH. As no other pathogenic variants were found in epilepsy-related genes, this FLNA variant likely caused West syndrome but with no PVNH.
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http://dx.doi.org/10.1038/s41439-020-00131-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713383PMC
December 2020

Whole exome sequencing of fetal structural anomalies detected by ultrasonography.

J Hum Genet 2020 Nov 3. Epub 2020 Nov 3.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

The objective of this study was to evaluate the efficacy of whole exome sequencing (WES) for the genetic diagnosis of cases presenting with fetal structural anomalies detected by ultrasonography. WES was performed on 19 cases with prenatal structural anomalies. Genomic DNA was extracted from umbilical cords or umbilical blood obtained shortly after birth. WES data were analyzed on prenatal phenotypes alone, and the data were re-analyzed after information regarding the postnatal phenotype was obtained. Based solely on the fetal phenotype, pathogenic, or likely pathogenic, single nucleotide variants were identified in 5 of 19 (26.3%) cases. Moreover, we detected trisomy 21 in two cases by WES-based copy number variation analysis. The overall diagnostic rate was 36.8% (7/19). They were all compatible with respective fetal structural anomalies. By referring to postnatal phenotype information, another candidate variant was identified by a postnatal clinical feature that was not detected in prenatal screening. As detailed phenotyping is desirable for better diagnostic rates in WES analysis, we should be aware that fetal phenotype is a useful, but sometimes limited source of information for comprehensive genetic analysis. It is important to amass more data of genotype-phenotype correlations, especially to appropriately assess the validity of WES in prenatal settings.
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http://dx.doi.org/10.1038/s10038-020-00869-8DOI Listing
November 2020

Efficient detection of copy-number variations using exome data: Batch- and sex-based analyses.

Hum Mutat 2021 Jan 11;42(1):50-65. Epub 2020 Nov 11.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

Many algorithms to detect copy number variations (CNVs) using exome sequencing (ES) data have been reported and evaluated on their sensitivity and specificity, reproducibility, and precision. However, operational optimization of such algorithms for a better performance has not been fully addressed. ES of 1199 samples including 763 patients with different disease profiles was performed. ES data were analyzed to detect CNVs by both the eXome Hidden Markov Model (XHMM) and modified Nord's method. To efficiently detect rare CNVs, we aimed to decrease sequencing biases by analyzing, at the same time, the data of all unrelated samples sequenced in the same flow cell as a batch, and to eliminate sex effects of X-linked CNVs by analyzing female and male sequences separately. We also applied several filtering steps for more efficient CNV selection. The average number of CNVs detected in one sample was <5. This optimization together with targeted CNV analysis by Nord's method identified pathogenic/likely pathogenic CNVs in 34 patients (4.5%, 34/763). In particular, among 142 patients with epilepsy, the current protocol detected clinically relevant CNVs in 19 (13.4%) patients, whereas the previous protocol identified them in only 14 (9.9%) patients. Thus, this batch-based XHMM analysis efficiently selected rare pathogenic CNVs in genetic diseases.
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http://dx.doi.org/10.1002/humu.24129DOI Listing
January 2021

De novo variants in CELF2 that disrupt the nuclear localization signal cause developmental and epileptic encephalopathy.

Hum Mutat 2021 Jan 10;42(1):66-76. Epub 2020 Nov 10.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan.

We report heterozygous CELF2 (NM_006561.3) variants in five unrelated individuals: Individuals 1-4 exhibited developmental and epileptic encephalopathy (DEE) and Individual 5 had intellectual disability and autistic features. CELF2 encodes a nucleocytoplasmic shuttling RNA-binding protein that has multiple roles in RNA processing and is involved in the embryonic development of the central nervous system and heart. Whole-exome sequencing identified the following CELF2 variants: two missense variants [c.1558C>T:p.(Pro520Ser) in unrelated Individuals 1 and 2, and c.1516C>G:p.(Arg506Gly) in Individual 3], one frameshift variant in Individual 4 that removed the last amino acid of CELF2 c.1562dup:p.(Tyr521Ter), possibly resulting in escape from nonsense-mediated mRNA decay (NMD), and one canonical splice site variant, c.272-1G>C in Individual 5, also probably leading to NMD. The identified variants in Individuals 1, 2, 4, and 5 were de novo, while the variant in Individual 3 was inherited from her mosaic mother. Notably, all identified variants, except for c.272-1G>C, were clustered within 20 amino acid residues of the C-terminus, which might be a nuclear localization signal. We demonstrated the extranuclear mislocalization of mutant CELF2 protein in cells transfected with mutant CELF2 complementary DNA plasmids. Our findings indicate that CELF2 variants that disrupt its nuclear localization are associated with DEE.
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http://dx.doi.org/10.1002/humu.24130DOI Listing
January 2021

Lenticular nuclei to thalamic ratio on PET is useful for diagnosis of GLUT1 deficiency syndrome.

Brain Dev 2021 Jan 30;43(1):69-77. Epub 2020 Jul 30.

Department of Radiological and Medical Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Purpose: To establish an objective method of [F]-fluorodeoxyglucose positron emission tomography (FDG-PET) that can assist in the diagnosis of glucose transporter 1 deficiency syndrome (GLUT1-DS).

Methods: FDG-PET was performed in 8 patients with a mean age of 12.5 years (range, 2-22 years) with GLUT1-DS. Their PET findings were compared with those of 45 controls with a mean age of 11.2 years (range, 2-21 years) by statistical parametric mapping (SPM12, Welcome Neurological Institute). The controls had epilepsy of unknown etiology and normal MRI findings. The age-adjusted ratios of mean radioactivities in regions of interest (ROIs) of bilateral lenticular nuclei, thalami, and the whole cerebral cortex were also measured. The sensitivities and specificities of the ratios for the differential diagnosis of GLUT1-DS were also determined.

Results: SPM showed significantly decreased uptake in bilateral thalami and increased uptake in bilateral lenticular nuclei in patients with GLUT1-DS. There were no areas in the cerebral cortex with significant differences between patients and controls. On ROI analysis, by setting the cut-off value of the age-adjusted lenticular nuclei/thalami radioactivity ratio to 1.54, patients with GLUT1-DS were differentiated from controls with sensitivity of 1.00 and specificity of 0.98.

Conclusion: The age-adjusted lenticular nuclei/thalami radioactivity ratio on PET can distinguish patients with GLUT1-DS from patients with epilepsy of unknown etiology with high sensitivity and specificity. It is important to pay attention to the metabolism of the lenticular nuclei and thalami on PET for the diagnosis of GLUT1-DS.
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http://dx.doi.org/10.1016/j.braindev.2020.07.001DOI Listing
January 2021

Prenatal clinical manifestations in individuals with variants.

J Med Genet 2020 Jul 30. Epub 2020 Jul 30.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan

Background: Variants in the type IV collagen gene () cause early-onset cerebrovascular diseases. Most individuals are diagnosed postnatally, and the prenatal features of individuals with variants remain unclear.

Methods: We examined in 218 individuals with suspected /2-related brain defects. Among those arising from variants, we focused on individuals showing prenatal abnormal ultrasound findings and validated their prenatal and postnatal clinical features in detail.

Results: Pathogenic variants were detected in 56 individuals (n=56/218, 25.7%) showing porencephaly (n=29), schizencephaly (n=12) and others (n=15). Thirty-four variants occurred de novo (n=34/56, 60.7%). Foetal information was available in 47 of 56 individuals, 32 of whom (n=32/47, 68.1%) had one or more foetal abnormalities. The median gestational age at the detection of initial prenatal abnormal features was 31 weeks of gestation. Only 14 individuals had specific prenatal findings that were strongly suggestive of features associated with variants. Foetal ventriculomegaly was the most common initial feature (n=20/32, 62.5%). Posterior fossa abnormalities, including Dandy-Walker malformation, were observed prenatally in four individuals. Regarding extrabrain features, foetal growth restriction was present in 16 individuals, including eight individuals with comorbid ventriculomegaly.

Conclusions: Prenatal observation of ventriculomegaly with comorbid foetal growth restriction should prompt a thorough ultrasound examination and gene testing should be considered when pathogenic variants are strongly suspected.
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http://dx.doi.org/10.1136/jmedgenet-2020-106896DOI Listing
July 2020

De Novo Truncating Variants in the Last Exon of SEMA6B Cause Progressive Myoclonic Epilepsy.

Am J Hum Genet 2020 04 12;106(4):549-558. Epub 2020 Mar 12.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan. Electronic address:

De novo variants (DNVs) cause many genetic diseases. When DNVs are examined in the whole coding regions of genes in next-generation sequencing analyses, pathogenic DNVs often cluster in a specific region. One such region is the last exon and the last 50 bp of the penultimate exon, where truncating DNVs cause escape from nonsense-mediated mRNA decay [NMD(-) region]. Such variants can have dominant-negative or gain-of-function effects. Here, we first developed a resource of rates of truncating DNVs in NMD(-) regions under the null model of DNVs. Utilizing this resource, we performed enrichment analysis of truncating DNVs in NMD(-) regions in 346 developmental and epileptic encephalopathy (DEE) trios. We observed statistically significant enrichment of truncating DNVs in semaphorin 6B (SEMA6B) (p value: 2.8 × 10; exome-wide threshold: 2.5 × 10). The initial analysis of the 346 individuals and additional screening of 1,406 and 4,293 independent individuals affected by DEE and developmental disorders collectively identified four truncating DNVs in the SEMA6B NMD(-) region in five individuals who came from unrelated families (p value: 1.9 × 10) and consistently showed progressive myoclonic epilepsy. RNA analysis of lymphoblastoid cells established from an affected individual showed that the mutant allele escaped NMD, indicating stable production of the truncated protein. Importantly, heterozygous truncating variants in the NMD(+) region of SEMA6B are observed in general populations, and SEMA6B is most likely loss-of-function tolerant. Zebrafish expressing truncating variants in the NMD(-) region of SEMA6B orthologs displayed defective development of brain neurons and enhanced pentylenetetrazole-induced seizure behavior. In summary, we show that truncating DNVs in the final exon of SEMA6B cause progressive myoclonic epilepsy.
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http://dx.doi.org/10.1016/j.ajhg.2020.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118575PMC
April 2020

Genetic abnormalities in a large cohort of Coffin-Siris syndrome patients.

J Hum Genet 2019 Dec 17;64(12):1173-1186. Epub 2019 Sep 17.

Department of Human Genetics, Graduate school of medicine, Yokohama City University, Yokohama, Japan.

Coffin-Siris syndrome (CSS, MIM#135900) is a congenital disorder characterized by coarse facial features, intellectual disability, and hypoplasia of the fifth digit and nails. Pathogenic variants for CSS have been found in genes encoding proteins in the BAF (BRG1-associated factor) chromatin-remodeling complex. To date, more than 150 CSS patients with pathogenic variants in nine BAF-related genes have been reported. We previously reported 71 patients of whom 39 had pathogenic variants. Since then, we have recruited an additional 182 CSS-suspected patients. We performed comprehensive genetic analysis on these 182 patients and on the previously unresolved 32 patients, targeting pathogenic single nucleotide variants, short insertions/deletions and copy number variations (CNVs). We confirmed 78 pathogenic variations in 78 patients. Pathogenic variations in ARID1B, SMARCB1, SMARCA4, ARID1A, SOX11, SMARCE1, and PHF6 were identified in 48, 8, 7, 6, 4, 1, and 1 patients, respectively. In addition, we found three CNVs including SMARCA2. Of particular note, we found a partial deletion of SMARCB1 in one CSS patient and we thoroughly investigated the resulting abnormal transcripts.
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http://dx.doi.org/10.1038/s10038-019-0667-4DOI Listing
December 2019

Comprehensive genetic analysis of 57 families with clinically suspected Cornelia de Lange syndrome.

J Hum Genet 2019 Oct 23;64(10):967-978. Epub 2019 Jul 23.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder with specific dysmorphic features. Pathogenic genetic variants encoding cohesion complex subunits and interacting proteins (e.g., NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major causes of CdLS. However, there are many clinically diagnosed cases of CdLS without pathogenic variants in these genes. To identify further genetic causes of CdLS, we performed whole-exome sequencing in 57 CdLS families, systematically evaluating both single nucleotides variants (SNVs) and copy number variations (CNVs). We identified pathogenic genetic changes in 36 out of 57 (63.2 %) families, including 32 SNVs and four CNVs. Two known CdLS genes, NIPBL and SMC1A, were mutated in 23 and two cases, respectively. Among the remaining 32 individuals, four genes (ANKRD11, EP300, KMT2A, and SETD5) each harbored a pathogenic variant in a single individual. These variants are known to be involved in CdLS-like. Furthermore, pathogenic CNVs were detected in NIPBL, MED13L, and EHMT1, along with pathogenic SNVs in ZMYND11, MED13L, and PHIP. These three latter genes were involved in diseases other than CdLS and CdLS-like. Systematic clinical evaluation of all patients using a recently proposed clinical scoring system showed that ZMYND11, MED13L, and PHIP abnormality may cause CdLS or CdLS-like.
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http://dx.doi.org/10.1038/s10038-019-0643-zDOI Listing
October 2019

Entire FGF12 duplication by complex chromosomal rearrangements associated with West syndrome.

J Hum Genet 2019 Oct 16;64(10):1005-1014. Epub 2019 Jul 16.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

Complex rearrangements of chromosomes 3 and 9 were found in a patient presenting with severe epilepsy, developmental delay, dysmorphic facial features, and skeletal abnormalities. Molecular cytogenetic analysis revealed 46,XX.ish der(9)(3qter→3q28::9p21.1→9p22.3::9p22.3→9qter)(RP11-368G14+,RP11-299O8-,RP11-905L2++,RP11-775E6++). Her dysmorphic features are consistent with 3q29 microduplication syndrome and inv dup del(9p). Trio-based WES of the patient revealed no pathogenic single nucleotide variants causing epilepsy, but confirmed a 3q28q29 duplication involving FGF12, which encodes fibroblast growth factor 12. FGF12 positively regulates the activity of voltage-gated sodium channels. Recently, only one recurrent gain-of-function variant [NM_021032.4:c.341G>A:p.(Arg114His)] in FGF12 was found in a total of 10 patients with severe early-onset epilepsy. We propose that the patient's entire FGF12 duplication may be analogous to the gain-of-function variant in FGF12 in the epileptic phenotype of this patient.
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http://dx.doi.org/10.1038/s10038-019-0641-1DOI Listing
October 2019

A common CHRNE mutation in Brazilian patients with congenital myasthenic syndrome.

J Neurol 2018 Mar 30;265(3):708-713. Epub 2018 Jan 30.

Departamento de Neurologia, Faculdade de Medicina da Universidade de São Paulo (FMUSP), Av. Enéas de Carvalho Aguiar, 255, 5o andar, sala 5084, Cerqueira César, São Paulo, 05403-900, Brazil.

The most common causes of congenital myasthenic syndromes (CMS) are CHRNE mutations, and some pathogenic allelic variants in this gene are especially frequent in certain ethnic groups. In the southern region of Brazil, a study found the c.130dupG CHRNE mutation in up to 33% of families with CMS. Here, we aimed to verify the frequency of this mutation among individuals with CMS in a larger cohort of CMS patients from different areas of Brazil and to characterize clinical features of these patients. Eighty-four patients with CMS, from 72 families, were clinically evaluated and submitted to direct sequencing of the exon 2 of CHRNE. The c.130dupG mutation was found in 32 patients (23 families), with 26 patients (19 families, 26.3%) in homozygosis, confirming its high prevalence in different regions of Brazil. Among the homozygous patients, the following characteristics were frequent: onset of symptoms before 2 years of age (92.3%), little functional restriction (92.3%), fluctuating symptoms (100%), ocular muscle impairment (96.1%), ptosis (100%), limb weakness (88.4%), response to pyridostigmine (100%), facial involvement (77%), and bulbar symptoms (70.8%). The pretest probability of finding at least one allele harbouring the c.130dupG mutation was 38.1%. Selecting only patients with impaired eye movement together with limb weakness and improvement with pyridostigmine, the probability increases to 72.2%. This clinical pre-selection of patients is likely a useful tool for regions where CHRNE mutations have a founder effect. In conclusion, the CHRNE mutation c.130dupG leads to fairly benign natural course of the disease with relative homogeneity.
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http://dx.doi.org/10.1007/s00415-018-8736-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115868PMC
March 2018

Cytotoxic edema at onset in West syndrome of unknown etiology: A longitudinal diffusion tensor imaging study.

Epilepsia 2018 02 8;59(2):440-448. Epub 2018 Jan 8.

Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Objective: To clarify longitudinal changes in white matter microstructures from the onset of disease in patients with West syndrome (WS) of unknown etiology.

Methods: Diffusion tensor imaging (DTI) was prospectively performed at onset and at 12 and 24 months old in 17 children with WS of unknown etiology. DTI was analyzed using tract-based spatial statistics (TBSS) and tract-specific analysis (TSA) of 13 fiber tracts, and fractional anisotropy (FA) and mean diffusivity (MD) were compared with those of 42 age-matched controls. Correlations of FA and MD with developmental quotient (DQ) at age 24 months were analyzed. Multiple comparisons were adjusted for using the false discovery rate (q-value).

Results: TBSS analysis at onset showed higher FA and lower MD in the corpus callosum and brainstem in patients. TSA showed lower MD in bilateral uncinate fasciculi (UF) (right: q < 0.001; left: q = 0.03) at onset in patients. TBSS showed a negative correlation between FA at onset and DQ in the right frontal lobe, whereas FA at 24 months old exhibited a positive correlation with DQ in the diffuse white matter. MD for bilateral UF at 24 months old on TSA correlated positively with DQ (q = 0.04, both).

Significance: These findings may indicate the existence of cytotoxic edema in the immature white matter and dorsal brainstem at onset, and subsequent alterations in the diffuse white matter in WS of unknown etiology. Microstructural development in the UF might play important roles in cognitive development in WS.
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http://dx.doi.org/10.1111/epi.13988DOI Listing
February 2018

MYRF is associated with encephalopathy with reversible myelin vacuolization.

Ann Neurol 2018 01 14;83(1):98-106. Epub 2018 Jan 14.

Department of Pediatrics, Aichi Medical University, Nagakute, Aichi, Japan.

Objective: Reversible myelin vacuolization is associated with variable conditions including mild encephalitis/encephalopathy with a reversible splenial lesion (MERS), which is characterized by mildly impaired consciousness and transient splenial lesion. Familial and/or recurrent cases with a clinical diagnosis of MERS suggest the presence of genetic factors.

Methods: We examined a family in which the proband presented with a history of recurrent encephalopathy with extensive but reversible cerebral myelin vacuolization and neurological symptoms similar to those of MERS spanning 3 generations. Whole-exome sequencing was performed in family members.

Results: Eight rare nonsynonymous single-nucleotide variants shared by all patients were identified. By filtering genes expressed in the corpus callosum, we identified a heterozygous c.1208A>G predicting p.Gln403Arg in the highly conserved DNA-binding domain in the myelin regulatory factor (MYRF) gene. We subsequently screened the coding regions of MYRF by Sanger sequencing in our cohort comprised of 33 sporadic cases with MERS and 3 cases in another family with extensive myelin vacuolization, and identified the same heterozygous c.1208A>G in all affected members in the second family. Luciferase assay revealed that transcriptional activity of the N-terminal region of MYRF was significantly diminished by introducing the c.1208A>G variant.

Interpretation: MYRF is a transcriptional regulator that is necessary for oligodendrocyte differentiation and myelin maintenance. Functional defects of MYRF are likely to be causally associated with encephalopathy with extensive myelin vacuolization. We propose the term "MYRF-related mild encephalopathy with reversible myelin vacuolization." Our findings provide a new perspective on the pathogenesis of myelin vacuolization. Ann Neurol 2018;83:98-106.
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http://dx.doi.org/10.1002/ana.25125DOI Listing
January 2018

Congenital Myasthenic Syndromes or Inherited Disorders of Neuromuscular Transmission: Recent Discoveries and Open Questions.

J Neuromuscul Dis 2017;4(4):269-284

Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.

Congenital myasthenic syndromes (CMS) form a heterogeneous group of rare diseases characterized by fatigable muscle weakness. They are genetically-inherited and caused by defective synaptic transmission at the cholinergic neuromuscular junction (NMJ). The number of genes known to cause CMS when mutated is currently 30, and the relationship between fatigable muscle weakness and defective functions is quite well-understood for many of them. However, some of the most recent discoveries in individuals with CMS challenge our knowledge of the NMJ, where the basis of the pathology has mostly been investigated in animal models. Frontier forms between CMS and congenital myopathy, which have been genetically and clinically identified, underline the poorly understood interplay between the synaptic and extrasynaptic molecules in the neuromuscular system. In addition, precise electrophysiological and histopathological investigations of individuals with CMS suggest an important role of NMJ plasticity in the response to CMS pathogenesis. While efficient drug-based treatments are already available to improve neuromuscular transmission for most forms of CMS, others, as well as neurological and muscular comorbidities, remain resistant. Taken together, the available pathological data point to physiological issues which remain to be understood in order to achieve precision medicine with efficient therapeutics for all individuals suffering from CMS.
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http://dx.doi.org/10.3233/JND-170257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701762PMC
July 2019

Intragenic deletion detected by whole-genome sequencing in congenital myasthenic syndromes.

Neurol Genet 2017 Jun 3;3(3):e152. Epub 2017 May 3.

Institute of Genetic Medicine (Y.A., A.T., T.E., P.J.L., A.R., H.L.), Newcastle University, UK; Division of Neurology (P.J.L.), Federal University of Parana, Brazil; Leibniz-Institut für Analytische Wissenschaften ISAS e.V. (A.R.), Germany; Department of Neurosciences and Mental Health (P.V.), University of Lisbon, Portugal; and Division of Molecular Genetics (H.I., H.K.), Fujita Health University, Japan.

Objective: To identify the genetic cause in a patient affected by ptosis and exercise-induced muscle weakness and diagnosed with congenital myasthenic syndromes (CMS) using whole-genome sequencing (WGS).

Methods: Candidate gene screening and WGS analysis were performed in the case. Allele-specific PCR was subsequently performed to confirm the copy number variation (CNV) that was suspected from the WGS results.

Results: In addition to the previously reported frameshift mutation c.1124_1127dup, an intragenic 6,261 bp deletion spanning from the 5' untranslated region to intron 2 of the gene was identified by WGS in the patient with CMS. The heterozygous deletion was suspected based on reduced coverage on WGS and confirmed by allele-specific PCR. The breakpoints had microhomology and an inverted repeat, which may have led to the development of the deletion during DNA replication.

Conclusions: We report a CMS case with identification of the breakpoints of the intragenic deletion using WGS analysis. This case illustrates that CNVs undetected by Sanger sequencing may be identified by WGS and highlights their relevance in the molecular diagnosis of a treatable neurologic condition such as CMS.
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http://dx.doi.org/10.1212/NXG.0000000000000152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415388PMC
June 2017

Fournier's gangrene during ACTH therapy.

Brain Dev 2017 May 20;39(5):435-438. Epub 2016 Dec 20.

Department of Pediatrics, Aichi Medical University, Japan.

Fournier's gangrene is an infectious necrotizing fasciitis of the perineal, genital, or perianal regions and is uncommon in children. Adrenocorticotropic hormone (ACTH) is effective for the treatment of infantile spasms; however, suppression of immune function is one of the major adverse effects of this approach. We encountered a 2-month-old boy with infantile spasms that had been treated with ACTH and had developed complicating Fournier's gangrene. Strangulation of a right inguinal hernia was observed after ACTH treatment. Although surgical repair was successful and no intestinal injuries were detected, swelling and discoloration of the right scrotum developed in association with pyrexia and a severe inflammatory response. A scrotal incision revealed pus with a putrid smell. The patient was subsequently diagnosed with Fournier's gangrene complicated by septic shock and disseminated intravascular coagulation. Extensive debridement and intensive care was performed. Enterobactor aerogenes, methicillin-resistant Staphylococcus aureus, and Enterococcus faecalis were isolated from the pus. Meropenem, teicoplanin, and clindamycin were administered to control the bacterial infection. The patient was discharged from the intensive care unit without any obvious neurological sequelae. Suppression of immune function associated with ACTH therapy may have been related to the development of Fournier's gangrene in this case.
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http://dx.doi.org/10.1016/j.braindev.2016.11.012DOI Listing
May 2017

Impaired Presynaptic High-Affinity Choline Transporter Causes a Congenital Myasthenic Syndrome with Episodic Apnea.

Am J Hum Genet 2016 09 25;99(3):753-761. Epub 2016 Aug 25.

Inserm U 1127, CNRS UMR 7225, Sorbonne Universités, UPMC Université Paris 06 UMR S 1127, Institut du Cerveau et de la Moelle épinière, ICM, 75013 Paris, France. Electronic address:

The neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMSs), a clinically and genetically heterogeneous group of rare diseases with fluctuating fatigable muscle weakness as the clinical hallmark. Whole-exome sequencing and Sanger sequencing in six unrelated families identified compound heterozygous and homozygous mutations in SLC5A7 encoding the presynaptic sodium-dependent high-affinity choline transporter 1 (CHT), which is known to be mutated in one dominant form of distal motor neuronopathy (DHMN7A). We identified 11 recessive mutations in SLC5A7 that were associated with a spectrum of severe muscle weakness ranging from a lethal antenatal form of arthrogryposis and severe hypotonia to a neonatal form of CMS with episodic apnea and a favorable prognosis when well managed at the clinical level. As expected given the critical role of CHT for multisystemic cholinergic neurotransmission, autonomic dysfunctions were reported in the antenatal form and cognitive impairment was noticed in half of the persons with the neonatal form. The missense mutations induced a near complete loss of function of CHT activity in cell models. At the human NMJ, a delay in synaptic maturation and an altered maintenance were observed in the antenatal and neonatal forms, respectively. Increased synaptic expression of butyrylcholinesterase was also observed, exposing the dysfunction of cholinergic metabolism when CHT is deficient in vivo. This work broadens the clinical spectrum of human diseases resulting from reduced CHT activity and highlights the complexity of cholinergic metabolism at the synapse.
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http://dx.doi.org/10.1016/j.ajhg.2016.06.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5011057PMC
September 2016

The effects of co-medications on lamotrigine clearance in Japanese children with epilepsy.

Brain Dev 2016 Sep 28;38(8):723-30. Epub 2016 Mar 28.

Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Purpose: Although it has been reported that some antiepileptic drugs have inducing or inhibiting effects on lamotrigine (LTG) clearance, whether they have the same effects in Asian epilepsy patients as in those in other countries has not been clarified, especially in children. The aim of this study was to determine the effects of co-medications on LTG clearance in Japanese children with epilepsy.

Methods: A total of 342 routine serum concentration measurements of LTG in 102 Japanese epilepsy patients under 20years of age were reviewed. The dose-corrected concentration (DCC) of LTG was calculated as [concentration]/[dose/(body weight)], and the DCC of LTG was compared by co-medication. The difference in the DCC of LTG was compared between patients with and without valproic acid (VPA) and between those with and without drugs inducing glucuronic acid conjugation (phenytoin (PHT), carbamazepine (CBZ), and phenobarbital (PB)).

Results: The DCC of LTG was significantly higher in patients on VPA and significantly lower in patients on drugs inducing glucuronic acid conjugation than in patients on LTG monotherapy. The DCC of LTG was significantly higher in patients on CBZ than in patients on PHT or PB. There was no correlation between the DCC of LTG and the concentration of VPA or metabolic inducers within the therapeutic range. Other antiepileptic drugs including clobazam, clonazepam, zonisamide, and levetiracetam had little effect on LTG concentration.

Conclusion: LTG concentration changes dramatically with concomitant antiepileptic drugs in Japanese children, as previously reported from other countries, and special attention is required. Although the dose of LTG should be adjusted when starting or discontinuing VPA or metabolic inducers, no adjustment is needed when changing the dose of VPA or metabolic inducers in the therapeutic range.
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http://dx.doi.org/10.1016/j.braindev.2016.03.004DOI Listing
September 2016

IntSplice: prediction of the splicing consequences of intronic single-nucleotide variations in the human genome.

J Hum Genet 2016 Jul 24;61(7):633-40. Epub 2016 Mar 24.

Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Precise spatiotemporal regulation of splicing is mediated by splicing cis-elements on pre-mRNA. Single-nucleotide variations (SNVs) affecting intronic cis-elements possibly compromise splicing, but no efficient tool has been available to identify them. Following an effect-size analysis of each intronic nucleotide on annotated alternative splicing, we extracted 105 parameters that could affect the strength of the splicing signals. However, we could not generate reliable support vector regression models to predict the percent-splice-in (PSI) scores for normal human tissues. Next, we generated support vector machine (SVM) models using 110 parameters to directly differentiate pathogenic SNVs in the Human Gene Mutation Database and normal SNVs in the dbSNP database, and we obtained models with a sensitivity of 0.800±0.041 (mean and s.d.) and a specificity of 0.849±0.021. Our IntSplice models were more discriminating than SVM models that we generated with Shapiro-Senapathy score and MaxEntScan::score3ss. We applied IntSplice to a naturally occurring and nine artificial intronic mutations in RAPSN causing congenital myasthenic syndrome. IntSplice correctly predicted the splicing consequences for nine of the ten mutants. We created a web service program, IntSplice (http://www.med.nagoya-u.ac.jp/neurogenetics/IntSplice) to predict splicing-affecting SNVs at intronic positions from -50 to -3.
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http://dx.doi.org/10.1038/jhg.2016.23DOI Listing
July 2016

SRSF1 and hnRNP H antagonistically regulate splicing of COLQ exon 16 in a congenital myasthenic syndrome.

Sci Rep 2015 Aug 18;5:13208. Epub 2015 Aug 18.

Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.

The catalytic subunits of acetylcholinesterase (AChE) are anchored in the basal lamina of the neuromuscular junction using a collagen-like tail subunit (ColQ) encoded by COLQ. Mutations in COLQ cause endplate AChE deficiency. An A-to-G mutation predicting p.E415G in COLQ exon 16 identified in a patient with endplate AChE deficiency causes exclusive skipping of exon 16. RNA affinity purification, mass spectrometry, and siRNA-mediated gene knocking down disclosed that the mutation disrupts binding of a splicing-enhancing RNA-binding protein, SRSF1, and de novo gains binding of a splicing-suppressing RNA-binding protein, hnRNP H. MS2-mediated artificial tethering of each factor demonstrated that SRSF1 and hnRNP H antagonistically modulate splicing by binding exclusively to the target in exon 16. Further analyses with artificial mutants revealed that SRSF1 is able to bind to degenerative binding motifs, whereas hnRNP H strictly requires an uninterrupted stretch of poly(G). The mutation compromised splicing of the downstream intron. Isolation of early spliceosome complex revealed that the mutation impairs binding of U1-70K (snRNP70) to the downstream 5' splice site. Global splicing analysis with RNA-seq revealed that exons carrying the hnRNP H-binding GGGGG motif are predisposed to be skipped compared to those carrying the SRSF1-binding GGAGG motif in both human and mouse brains.
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http://dx.doi.org/10.1038/srep13208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539547PMC
August 2015

Seizure characteristics of epilepsy in childhood after acute encephalopathy with biphasic seizures and late reduced diffusion.

Epilepsia 2015 Aug 29;56(8):1286-93. Epub 2015 Jun 29.

Department of Pediatrics, Nagoya University Graduate School of Medicine, Aichi, Japan.

Objective: The aim of this study was to clarify characteristics of post-encephalopathic epilepsy (PEE) in children after acute encephalopathy with biphasic seizures and late reduced diffusion (AESD), paying particular attention to precise diagnosis of seizure types.

Methods: Among 262 children with acute encephalopathy/encephalitis registered in a database of the Tokai Pediatric Neurology Society between 2005 and 2012, 44 were diagnosed with AESD according to the clinical course and magnetic resonance imaging (MRI) findings and were included in this study. Medical records were reviewed to investigate clinical data, MRI findings, neurologic outcomes, and presence or absence of PEE. Seizure types of PEE were determined by both clinical observation by pediatric neurologists and ictal video-electroencephalography (EEG) recordings.

Results: Of the 44 patients after AESD, 10 (23%) had PEE. The period between the onset of encephalopathy and PEE ranged from 2 to 39 months (median 8.5 months). Cognitive impairment was more severe in patients with PEE than in those without. Biphasic seizures and status epilepticus during the acute phase of encephalopathy did not influence the risk of PEE. The most common seizure type of PEE on clinical observation was focal seizures (n = 5), followed by epileptic spasms (n = 4), myoclonic seizures (n = 3), and tonic seizures (n = 2). In six patients with PEE, seizures were induced by sudden unexpected sounds. Seizure types confirmed by ictal video-EEG recordings were epileptic spasms and focal seizures with frontal onset, and all focal seizures were startle seizures induced by sudden acoustic stimulation. Intractable daily seizures remain in six patients with PEE.

Significance: We demonstrate seizure characteristics of PEE in children after AESD. Epileptic spasms and startle focal seizures are common seizure types. The specific seizure types may be determined by the pattern of diffuse subcortical white matter injury in AESD and age-dependent reorganization of the brain network.
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http://dx.doi.org/10.1111/epi.13068DOI Listing
August 2015

Hydrogen ameliorates pulmonary hypertension in rats by anti-inflammatory and antioxidant effects.

J Thorac Cardiovasc Surg 2015 Sep 22;150(3):645-54.e3. Epub 2015 May 22.

Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Objective: The pathogenesis of pulmonary arterial hypertension (PAH) involves reactive oxygen species and inflammation. Beneficial effects of molecular hydrogen, which exerts both anti-inflammatory and antioxidative effects, have been reported for various pathologic conditions. We therefore hypothesized that molecular hydrogen would improve monocrotaline (MCT)-induced PAH in rats.

Methods: Nineteen male Sprague-Dawley rats (body weight: 200-300 g) were divided into groups, receiving: (1) MCT + hydrogen-saturated water (group H); (2) MCT + dehydrogenized water (group M); or (3) saline + dehydrogenized water (group C). Sixteen days after substance administration, we evaluated hemodynamics, harvested the lungs and heart, and performed morphometric analysis of the pulmonary vasculature. Macrophage infiltration, antiproliferating cell nuclear antigen-positive cells, 8-hydroxy-deoxyguanosine (8-OHdG)-positive cells, and expressions of phosphorylated signal transducers and activators of transcription-3 (STAT3) and nuclear factor of activated T-cells (NFAT) were evaluated immunohistochemically. Stromal cell-derived factor-1 and monocyte chemoattractant protein-1 expressions were evaluated by quantitative reverse-transcription polymerase chain reaction.

Results: Pulmonary arterial hypertension was significantly exacerbated in group M compared to group C, but was significantly improved in group H. Vascular density was significantly reduced in group M, but not in group H. Adventitial macrophages, antiproliferating cell nuclear antigen - and 8-OHdG-positive cells, and stromal cell-derived factor-1 and monocyte chemoattractant protein-1 expressions were significantly increased in group M, but improved in group H. Expressions of phosphorylated STAT3 and NFAT were up-regulated in group M, but improved in group H.

Conclusions: Molecular hydrogen ameliorates MCT-induced PAH in rats by suppressing macrophage accumulation, reducing oxidative stress and modulating the STAT3/NFAT axis.
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http://dx.doi.org/10.1016/j.jtcvs.2015.05.052DOI Listing
September 2015

Congenital myasthenic syndrome in Japan: ethnically unique mutations in muscle nicotinic acetylcholine receptor subunits.

Neuromuscul Disord 2015 Jan 10;25(1):60-9. Epub 2014 Sep 10.

Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan. Electronic address:

Congenital myasthenic syndromes (CMS) are caused by mutations in genes expressed at the neuromuscular junction. Most CMS patients have been reported in Western and Middle Eastern countries, and only four patients with COLQ mutations have been reported in Japan. We here report six mutations in acetylcholine receptor (AChR) subunit genes in five Japanese patients. Five mutations are novel, and one mutation is shared with a European American patient but with a different haplotype. Among the observed mutations, p.Thr284Pro (p.Thr264Pro according to the legacy annotation) in the epsilon subunit causes a slow-channel CMS. Five other mutations in the delta and epsilon subunits are splice site, frameshift, null, or missense mutations causing endplate AChR deficiency. We also found a heteroallelic p.Met465Thr in the beta subunit in another patient. p.Met465Thr, however, was likely to be polymorphism, because single channel recordings showed mild shortening of channel openings without affecting cell surface expression of AChR, and the minor allelic frequency of p.Met465Thr was 5.1% in the Japanese population. Lack of shared mutant alleles between the Japanese and the other patients suggests that most mutations described here are ethnically unique or de novo in each family.
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http://dx.doi.org/10.1016/j.nmd.2014.09.002DOI Listing
January 2015

Mutations in the C-terminal domain of ColQ in endplate acetylcholinesterase deficiency compromise ColQ-MuSK interaction.

Hum Mutat 2013 Jul 19;34(7):997-1004. Epub 2013 Apr 19.

Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is mostly composed of an asymmetric form in which three tetramers of catalytic AChE subunits are linked to a triple helical collagen Q (ColQ). Mutations in COLQ cause endplate AChE deficiency. We report three patients with endplate AChE deficiency with five recessive COLQ mutations. Sedimentation profiles showed that p.Val322Asp and p.Arg227X, but not p.Cys444Tyr, p.Asp447His, or p.Arg452Cys, inhibit formation of triple helical ColQ. In vitro overlay of mutant ColQ-tailed AChE on muscle sections of Colq(-/-) mice revealed that p.Cys444Tyr, p.Asp447His, and p.Arg452Cys in the C-terminal domain (CTD) abrogate anchoring ColQ-tailed AChE to the NMJ. In vitro plate-binding assay similarly demonstrated that the three mutants inhibit binding of ColQ-tailed AChE to MuSK. We also confirmed the pathogenicity of p.Asp447His by treating Colq(-/-) mice with adeno-associated virus serotype 8 carrying mutant COLQ-p.Asp447His. The treated mice showed no improvement in motor functions and no anchoring of ColQ-tailed AChE at the NMJ. Electroporation of mutant COLQ harboring p.Cys444Tyr, p.Asp447His, and p.Arg452Cys into anterior tibial muscles of Colq(-/-) mice similarly failed to anchor ColQ-tailed AChE at the NMJ. We proved that the missense mutations in ColQ-CTD cause endplate AChE deficiency by compromising ColQ-MuSK interaction at the NMJ.
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http://dx.doi.org/10.1002/humu.22325DOI Listing
July 2013

[Clinical features of congenital myasthenic syndrome in Japan].

No To Hattatsu 2012 Nov;44(6):450-4

Department of Child Neurology, National Center Hospital of Neurology and Psychiatry, Kodaira, Tokyo.

Objective: We examined the clinical and neurophysiological features of Japanese patients with congenital myasthenic syndrome (CMS).

Method: Subjects were five patients who were diagnosed with CMS on the basis of clinical course, repetitive nerve stimulation (RNS), and genetic analysis.

Results: Four patients manifested motor retardation within one year of birth, while one manifested motor intolerance at three years of age. The most characteristic symptom observed in all the patients was fluctuating muscle weakness, which varied on a daily basis or continued for several days after the late infancy period. Only one patient manifested daily fluctuation of muscle weakness. RNS of the accessory nerve evoked a decrementing response in three patients who were examined;however, RNS of the median, ulnar, and tibial nerves (one patient each) did not evoke such responses. After the edrophonium chloride test, no improvement was seen even if the patients manifested ptosis. For judgment of this test, improvement in decrementing rate observed while performing RNS was useful. All five patients who were administered medication based on the results of genetic analysis demonstrated an improvement in their symptoms.

Conclusion: We suggest that CMS can be diagnosed based on careful examination and electrophysiological results. CMS is a treatable disorder, and therefore, correct diagnosis is important.
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November 2012

Long-term follow-up of patients with benign partial epilepsy in infancy.

Epilepsia 2006 Jan;47(1):181-5

Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Purpose: The aim of this study was to investigate the long-term outcome of children with benign partial epilepsy in infancy (BPEI).

Methods: A telephone-interview survey using a structured questionnaire was conducted with patients who were diagnosed as having possible BPEI at age 2 years and who were 8 years or older at the time of the survey. The data from 39 of 48 patients were available. The median age at the time of the survey was 11.3 years; 18 boys and 21 girls were included.

Results: Three patients had a recurrence of unprovoked seizure beyond age 2 years. Four patients had cognitive problems (mild mental retardation in three and Asperger syndrome in one). An association of paroxysmal kinesigenic choreoathetosis was observed in three patients, and another three had experienced seizures associated with mild gastroenteritis. Major behavioral problems were not recognized in any patients. Four patients were excluded from having definite BPEI at age 5 years, and another two were excluded for having definite BPEI at the last follow-up. Eventually, 33 of 39 patients were categorized as having definite BPEI beyond 8 years of age.

Conclusions: A large majority of patients diagnosed as possibly having BPEI at age 2 years did not have a recurrence of unprovoked seizures and cognitive problems beyond 8 years of age.
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http://dx.doi.org/10.1111/j.1528-1167.2006.00385.xDOI Listing
January 2006